Arachidonic acid is released from membrane phospholipids by the enzyme phospholipase A2. This highly unsaturated fatty acid is metabolized through one of three pathways: the cyclooxygenases (“COX”), the lipoxygenases (“LOX”), and the cytochrome P450 pathway. COX enzymes convert arachidonic acid to the prostaglandin endoperoxide PGH2, from which other prostaglandins are formed. See, e.g., Meade et al., J Biol. Chem. 274(12):8328-34 (1999). A number of drugs inhibit the action of either the COX or the LOX enzymes.
Non-steroidal anti-inflammatory drugs (“NSAIDs”) are thought to exert their effect by primarily inhibiting the COX enzymes, thereby blocking production of prostaglandins and thromboxanes. The beneficial effects are thought to come from directly inhibiting PGE2 and 5 other major metabolites. The titers of over 70 ‘by stander’ eicosanoids, however, also are altered. Some of these eicosanoids are critical for normal physiology; thus, it has long been a goal to reduce the activity of COX enzymes associated with an over-active inflammatory response while not disturbing the normal functioning of approximately one-third of the carbon flow from arachidonic acid through the COX enzymes to prostaglandins and thromboxanes necessary for normal physiology.
Elucidation of the two COX isoforms COX-1 and COX-2 gave rise to the concept that the constitutive enzyme COX-1 was responsible for the production of prostaglandins and thromboxanes with homeostatic functions in platelets and such tissues as the stomach and kidney, while COX-2, the inducible enzyme, was reported for the production of prostaglandins involved in inflammation. See, e.g., Seibert and Masferrer, Receptor, 4:17 (1994). Accordingly, it was thought that the therapeutic effects of NSAIDs were attributable to the inhibition of COX-2, while the inhibition of COX-1 accounted for the adverse affects associated with these drugs. See, e.g., Lichtenstein et al., Arthritis Rheum 38:5 (1995). This is actually an oversimplification, because COX-2 is expressed constitutively in the brain, airway epithelium, prostate, and macula densa of the kidney.
Although NSAIDs are highly effective, their use is associated with a number of adverse effects such as gastrointestinal ulceration and bleeding, inhibition of platelet aggregation, and adverse changes in renal blood flow. Gastrointestinal toxicity associated with chronic NSAID used is estimated to result in more than 100,000 hospitalizations and 16,000 deaths per year in the U.S. alone. See, e.g., Singh and Tridafilopoulos, J. Rheumatol. Supp., 56:18 (1999). The side effects of such gastritis were thought to occur through COX-1 inhibition. This led to the development of selective COX-2 inhibitors to block the pro-inflammatory mediators and reduce the side effects of NSAIDs. These drugs (also known as “coxibs”) are designed to specifically inhibit COX-2 and to have little effect on COX-1 activity across the therapeutic range. Examples of COX-2 inhibitors include celecoxib (Celebrex®, Pharmacia, Peapack, N.J.), refecoxib (Vioxx®, Merck, Whitehouse Station, N.J.), valdecoxib (Bextra®, Pfizer, New York, N.Y.), lumiracoxib (Prexige®, Novartis International AG, Basel, Switzerland), and etoricoxib (Arcoxia®, Merck, Whitehouse Station, N.J.).
Recent studies have challenged the hypothesis that COX-1 plays no role in inflammation and that COX-2 is the only isoform responsible for the synthesis of pro-inflammatory prostaglandins. In the rat carrageenin-induced pleurisy model, drugs more selective for COX-2 inhibition attenuated inflammation over wider time frame than selective COX-1 inhibitors, thus suggesting a role of COX-2 in this model. There is also increasing evidence that the inhibition of COX-2 delays the resolution of inflammation. Gilroy et al., FASEB J 15:288 (2001); Gilroy et al., Am J Physiol Cell Physiol 281:C188 (2001).
Despite their efficacy in the treatment of arthritic disease and chronic pain, NSAIDs are limited by their adverse drug interactions with anti-coagulants (e.g. warfarin) and anti-hypertensive drugs (e.g. angiotensin converting enzyme (“ACE”) inhibitors). NSAIDs increase gastric irritation and erosion of the protective lining of the stomach, assisting in the formation of gastrointestinal bleeding. Additionally, NSAIDs decrease the cohesive properties of platelets necessary in clot formation; thus, the addition of warfarin can lead to coughing up blood, bleeding gums, and blood in urine and stool. A few patients treated with an angiotensin converting enzyme (“ACE”) inhibitor and rofecoxib (Vioxx®), a selective COX-2 inhibitor, have developed serious renal problems that have led to severe hyperkalemia and death. Hay et al., J Emerg Med 22:349 (2002). Additionally, NSAIDs, especially aspirin, have been implicated in Reye's syndrome and in inducing asthmatic attacks in patients with asthma. This effect illustrates one of the problems in inhibiting either or both of the COX enzymes, because this inhibition shuttles arachidonate into other pathways in the arachidonate cascade, predominantly thought to be the LOX pathway. Increasing the flow of arachidonate through this pathway may itself result in undesirable side effects, including the production of pro-inflammatory mediators by the LOX enzymes, particularly 5-LOX.
Recently, some COX-2 inhibitors have been associated with higher risks for heart attacks or stroke. The associations are believed to be dose dependent. It has now been shown that selective COX-2 inhibitors depress PGI2 without concomitant inhibition of TXA2, which can result in an augmented response to thrombotic and hypertensive stimuli and acceleration of atherogenesis.
Epoxide hydrolases (“EHs”) are enzymes that add water to epoxides, resulting in their corresponding 1,2-diols (Hammock, B. D. et al., in Comprehensive Toxicology: Biotransformation (Elsevier, New York), pp. 283-305 (1997); Oesch, F. Xenobiotica 3:305-340 (1972)). Four principal EH's are known: leukotriene epoxide hydrolase, cholesterol epoxide hydrolase, microsomal EH (“mEH”), and soluble EH (“sEH,” previously called “cytosolic EH”). The leukotriene EH acts on leukotriene A4, whereas the cholesterol EH hydrates compounds related to the 5,6-epoxide of cholesterol (Nashed, N. T., et al., Arch. Biochem. Biophysics., 241:149-162 (1985); Finley, B. and B. D. Hammock, Biochem. Pharmacol., 37:3169-3175 (1988)). The microsomal epoxide hydrolase metabolizes monosubstituted, 1,1-disubstituted, cisital-1,2-disubstituted epoxides and epoxides on cyclic systems epoxides to their corresponding diols. Because of its broad substrate specificity, this enzyme is thought to play a significant role in ameliorating epoxide toxicity. Reactions of detoxification typically decrease the hydrophobicity of a compound, resulting in a more polar and thereby excretable substance.
Soluble EH is only very distantly related to mEH and hydrates a wide range of epoxides not on cyclic systems. In contrast to the role played in the degradation of potential toxic epoxides by mEH, sEH is believed to play a role in the formation or degradation of endogenous chemical mediators. For instance, cytochrome P450 epoxygenase catalyzes NADPH-dependent enatioselective epoxidation of arachidonic acid to four optically active cis-epoxyeicosantrienoic acids (“EETs”) (Karara, A., et al., J. Biol. Chem., 264:19822-19877 (1989)). Soluble epoxide hydrolase has been shown in vivo to convert these compounds with regio- and enantiofacial specificity to the corresponding vic-dihydroxyeicosatrienoic acids (“DHETs”). Both liver and lung cytosolic fraction hydrolyze 14,15-EET, 8,9-EET and 11,12-EET, in that order of preference. The 5,6 EET is hydrolyzed more slowly. Purified sEH selects 8S,9R- and 14R,15S-EET over their enantiomers as substrates. Studies have revealed that EETs and their corresponding DHETs exhibit a wide range of biological activities. Some of these activities include involvements in luteinizing hormone-releasing hormone, stimulation of luteinizing hormone release, inhibition of Na+/K+ ATPase, vasodilation of coronary artery, mobilization of Ca2+ and inhibition of platelet aggregation.